Previous studies that defined sex dependency of opioid antinociception were not poised to reveal relevant mechanistic underpinnings and biological substrates. The present application proposes to establish a causal association between sex-dependent pharmacological characteristics of spinal opioid antinociception and (1) the sexual dimorphic nature of spinal opioid receptors and (2) the sex-dependent release of spinal opioids by intrathecal (i.t.) morphine. Hypothesized sexual dimorphic relationships among spinal ?-opioid receptors (MOR), endomorphin 2 (EM2), ?-opioid receptors (KOR) and dynorphin 1-17 (Dyn) are supported by substantial preliminary data, which is undergirded by our previous demonstrations that i.t. morphine elicits predominantly MOR-coupled spinal antinociception in males, whereas in females i.t. morphine recruits a more inclusive integrative system that requires concomitant activity of spinal MOR and KOR. Sexual dimorphic expression levels of spinal MOR splice variants and MOR KOR heterodimers are postulated to be molecular underpinnings of the female phenotypic response to spinal morphine. In parallel with the postulated sexually dimorphic organization of spinal opioid receptors, we hypothesize that the sex-dependent regulation of the release of and analgesic responsiveness to spinal opioids are integral component of sex-dependent spinal opioid antinociception. Specifically, Aim 1 will test the hypothesis that the antinociception produced by i.t. morphine, requires the release of EM2 from the spinal cord of males, but not females. Aim 2 will test the hypothesis that i.t. EM2 elicits greater antinociception in males vs. females, which exacerbates the consequences of the sex-dependent release of spinal EM2 by i.t. morphine. We postulate that this is mediated, at lest in part, by the sex-dependent expression of spinal MOR splice variants. Validation that release of spinal EM2 by i.t. morphine is a prerequisite for the spinal antinociception it produces in males, but not females, would not fully reveal the sex-dependent landscape that underlies spinal morphine antinociception. To do so requires knowledge of the analgesic substrates recruited by i.t. morphine in females. Accordingly, Aim 3 will test the hypothesis that in females, i.t. morphine analgesia is mediated via the release of Dyn, not EM2. Aim 4 will test the hypothesis that the requirement in females for the concomitant activation of spinal MOR and KOR for spinal morphine antinociception to be manifest reflects the presence of significantly higher expression levels of MOR KOR heterodimers in spinal cord of females vs. males. We will pursue these interrelated Aims using a multi-dimensional approach that integrates behavioral, pharmacological, molecular and immuno-based biochemical levels of analyses. These will be used as complementary measures to cross validate findings. Since pain and addiction share common substrates, expected outcomes will reveal novel drug targets that are not only relevant to optimizing pain management in men and women but also to treating opioid withdrawal and relapse in a sex-dependent fashion. PUBLIC HEALTH RELEVANCE: This application will establish sexual dimorphic functional relationships among spinal ?- and ?-opioid receptors and the endogenous opioids endomorphin 2 and dynorphin 1-17. This will provide a rationale framework for understanding the sex divide in pain processing and its differential regulation in men vs. women.. Since pain and addiction share common substrates, expected outcomes will reveal novel drug targets and mechanisms that are not only relevant to the sex-dependent optimization of pain management but also to treating opioid withdrawal and relapse in women as well as men.